Biomedical Engineering Reference
In-Depth Information
2
How to Build an
SHG Apparatus
Riccardo Cicchi
National Institute of
Optics—National Research
Council (INO— CNR)
Leonardo Sacconi
European Laboratory for
Non-Linear Spectroscopy
(LENS)
National Institute of
Optics—National Research
Council (INO—CNR)
2.1 Mechanical System ............................................................................. 21
Optical Table • Microscope Stand
2.2 Laser Scanning System ......................................................................25
Basic Principles of Laser Scanning • Laser Source • Laser
Power Adjustment • Scanning Head • Microscope
Objective Lens • Polarization Scanning • A Multifocal SHG
Microscope • An SHG Holographic Microscope
2.3 The Detection System.........................................................................39
Optical Detection Modalities • Detection Geometry • Optical
Filtering • Detectors
2.4 Optical Scheme of a Typical SHG Microscope...............................44
Forward Detection • Backward Detection
References........................................................................................................47
Francesco Vanzi
European Laboratory for
Non-Linear Spectroscopy
(LENS)
University of Florence
Francesco S. Pavone
European Laboratory for
Non-Linear Spectroscopy
(LENS)
University of Florence
Over recent years, second-harmonic generation (SHG) microscopy has been well established as a micro-
scopic imaging modality used in biophysics, biomedical optics, and biophotonics applications. It has
already been widely demonstrated that SHG is a powerful technique for both cellular and tissue imag-
ing. However, a commercial microscope exclusively dedicated to SHG imaging is still lacking. Very
often, SHG microscopes used in laboratory research are adapted from commercial (confocal or multi-
photon) microscopes. However, for some applications,
ex novo
design is required to fit the technical
specifications of the experiments to be performed. Several technical and experimental aspects have to be
considered when designing a custom SHG microscope. This chapter aims to provide a technical guide to
the researchers who want to build their own custom SHG microscope. This chapter is divided into three
main parts. Starting from the possible solutions to be adopted for the mechanical system (Section 2.1),
we then focus our attention on the most critical points of the experimental setup: the scanning (Section
2.2) and the detection (Section 2.3) systems. In each part of this chapter, we describe the most common
configurations used in an experimental setup, highlighting their advantages and disadvantages, and
focusing the attention of the reader on the most crucial aspects to be considered during both design
and development. Finally (Section 2.4), we provide a short description of typical optical schemes used
in SHG microscopy.
2.1 Mechanical System
In this section, we describe the mechanical elements required for building an SHG microscope. Starting
from the optical table, we then focus our attention on the different solutions adaptable for the micro-
scope stand, highlighting advantages and drawbacks of custom solutions in comparison to commer-
cially available systems.
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